Enzymes are large proteins that catalyze reactions in living cells. Enzymes build up or tear down other molecules. For example, enzymes catalyze the synthesis of fat from fatty acids, form complex sugars from glucose and fructose, and aid in the formation of other proteins from amino acids. Enzymes also reverse the build-up process by breaking down more complex structures. Enzymes are generally specific to certain substrates for their reactions. For example, an individual enzyme may catalyze the reaction where only one substrate is involved or it may act on a group of related substrates.
Protein kinases are enzymes which catalyze the transfer of phosphorous from adenosine triphosphate (ATP), or guanosine triphosphate (GTP) to the targeted protein to yield a phosphorylated protein and adenosine diphosphate (ADP) or guanosine diphosphate (GDP), respectively. ATP or GTP is first hydrolyzed to form ADP or GDP and inorganic phosphate. The inorganic phosphate is then attached to the targeted protein. The protein substrate which is targeted by kinases may be a structural protein, found in membrane material such as a cell wall, or another enzyme which is a functional protein.
It is estimated about three to four percent of the human genome contains transcription information for the formation of protein kinases. Currently, there are about up to 400 known different protein kinases. However, because three to four percent of the human genome is a code for the formation of protein kinases, there may be many thousands of distinct and separate kinases in the human body.
Due to their physiological relevance, variety and ubiquitousness, protein kinases have become one of the most important and widely studied family of enzymes in biochemical and medical research. Studies have shown that protein kinases are key regulators of many cell functions, including signal transduction, transcriptional regulation, cell motility, and cell division. Several oncogenes have also been shown to encode protein kinases, suggesting that kinases play a role in oncogenesis.
Protein kinases are often divided into two groups based on the amino acid residue they phosphorylate. The first group, called serine/threonine kinases (PSTK), includes cyclic AMP and cyclic GMP dependent protein kinases, calcium and phospholipid dependent protein kinase, calcium and calmodulin-dependent protein kinases, casein kinases, cell division cycle protein kinases and others. These kinases are usually cytoplasmic or associated with the particulate fractions of cells, possibly by anchoring proteins. Aberrant protein serine/threonine kinase activity has been implicated or is suspected in a number of pathologies such as rheumatoid arthritis, psoriasis, septic shock, bone loss, many cancers and other proliferative diseases. Accordingly, serine/threonine kinases and the signal transduction pathways which they are part of are important targets for drug design.
The second group of kinases, called tyrosine kinases, phosphorylate tyrosine residues. They are present in much smaller quantities but also play an equally important role in cell regulation. These kinases include several receptors for molecules such as growth factors and hormones, including epidermal growth factor receptor, insulin receptor, platelet derived growth factor receptor and others. Studies have indicated that many tyrosine kinases are transmembrane proteins with their receptor domains located on the outside of the cell and their kinase domains on the inside. These transmembrane tyrosine kinases are called receptor tyrosine kinases (as opposed to non-receptor tyrosine kinases.)
Angiogenesis, the formation of new vessels from the pre-existing primary plexus, occurs through several processes: vascular sprouting, branching and pruning, and the differential growth of blood vessels to form mature vascular networks. Although the cellular and molecular mechanisms underlying angiogenesis are still poorly understood, signals are transduced through endothelial cell specific molecules, particularly receptor tyrosine kinases (RTKs) and their ligands. Vascular endothelial growth factor receptors (VEGFRs), Tie receptors, and Eph receptors are primarily involved in the process of angiogenesis. Consequently, targeting of pro-angiogenic pathways is a strategy being widely pursued in order to provide new therapeutics for cancers.
Vascular endothelial growth factor (VEGF) is a peptide mitogenic for endothelial cells in vitro and stimulates angiogenic responses in vivo. VEGF has also been linked to inappropriate angiogenesis (Pinedo, H. M. et al The Oncologist, Vol. 5, No. 90001, 1-2, Apr. 2000). VEGFR(s), which are receptors for VEGF, are receptor tyrosine kinases that catalyze the phosphorylation of specific tyrosyl residues in proteins involved in the regulation of cell growth and differentiation. (A. F. Wilks, Progress in Growth Factor Research, 1990, 2, 97-111; S. A. Courtneidge, Dev. Supp. I, 1993, 57-64; J. A. Cooper, Semin. Cell Biol., 1994, 5(6), 377-387; R. F. Paulson, Semin. Immunol., 1995, 7(4), 267-277; A. C. Chan, Curr. Opin. Immunol., 1996, 8(3), 394-401). Three protein tyrosine kinase (PTK) receptors for VEGF have been identified: VEGFR-1 (Flt-1); VEGFR-2 (Flk-1 or KDR) and VEGFR-3 (Flt-4). These receptors are involved in angiogenesis and participate in signal transduction (Mustonen, T. et al J. Cell Biol. 1995:129:895-898). Of particular interest is VEGFR-2, which is a transmembrane receptor PTK expressed primarily in endothelial cells. Activation of VEGFR-2 by VEGF is a critical step in the signal transduction pathway that initiates tumor angiogenesis. VEGF expression may be constitutive to tumor cells and can also be upregulated in response to certain stimuli. One such stimuli is hypoxia, where VEGF expression is upregulated in both tumor and associated host tissues. The VEGF ligand activates VEGFR-2 by binding with its extracellular VEGF binding site. This leads to receptor dimerization of VEGFRs and autophosphorylation of tyrosine residues at the intracellular kinase domain of VEGFR-2. The kinase domain operates to transfer a phosphate from ATP to the tyrosine residues, thus providing binding sites for signaling proteins downstream of VEGFR-2 leading ultimately to initiation of angiogenesis (McMahon, G., The Oncologist, Vol. 5, No. 90001, 3-10, Apr. 2000).
Angiopoieten 1 (Ang1), a ligand for the endothelium-specific receptor tyrosine kinase TIE-2, is a novel angiogenic factor (Davis et al, Cell, 1996, 87:1161-1169; Partanen et al, Mol. Cell. Biol, 12:1698-1707 (1992); U.S. Pat. Nos. 5,521,073; 5,879,672; 5,877,020; and 6,030,831). The acronym TIE represents “tyrosine kinase containing Ig and EGF homology domains”. TIE is used to identify a class of receptor tyrosine kinases, which are exclusively expressed in vascular endothelial cells and early hemopoietic cells. Typically, TIE receptor kinases are characterized by the presence of an EGF-like domain and an immunoglobulin (IG) like domain, which consists of extracellular folding units, stabilized by intra-chain disulfide bonds (Partanen et al Curr. Topics Microbiol. Immunol., 1999, 237:159-172). Unlike VEGF, which functions during the early stages of vascular development, Ang1 and its receptor TIE-2 function in the later stages of vascular development, i.e., during vascular remodeling (remodeling refers to formation of a vascular lumen) and maturation (Yancopoulos et al, Cell, 1998, 93:661-664; Peters, K. G., Circ. Res., 1998, 83(3):342-3; Suri et al, Cell 87, 1171-1180 (1996)).
EphB4 is another receptor tyrosine kinase originally described in J Biol Chem as HTK (1994 May 13; 269(19):14211-8) by Bennett B D et al. The recent observation of vascular defects in ephrin-B2 and EphB4 knockout mice strongly suggests that the interaction between the ephrin-B2 ligand and its cognate EphB4 receptor defines the boundaries of arterial-venous domains. Ephrin-B2 ligands are broadly expressed in several other nonvascular tissues such as mesenchymal cells adjacent to vascular endothelial cells, but EphB4 receptors are uniquely localized in vascular endothelial cells. Not only EphB4 receptors are activated by their respective ephrin-B2 ligands, which are also transmembrane proteins, but EphB4 receptors also activate their ephrin-B2 ligands. Embryos heterozygous for EphB4 allele do not show any apparent defects in comparison to wild type. However, homozygous embryos display cardiovascular defects from endothelial cell growth retardation and arrested heart development, and embryonic lethality with high incidence. These results clearly indicate EphB4 signaling pathway plays an essential role in vasculogenesis, angiogenesis and vessel maturation, and these events are also inextricably linked to cancer and atherosclerosis.
It is also known non-receptor tyrosine kinases which are located intracellularly are involved in the transmission of biochemical signals such as those that influence tumor cell motility, dissemination and invasiveness and subsequently metastatic tumor growth. Various classes of non-receptor tyrosine kinases are known including the Src family such as Src, Lyn, Fyn and Yes tyrosine kinases, the Abl family such as Abl and Arg and the Jak family such as Jak1 and Tyk 2.
It is known that the Src family of non-receptor tyrosine kinases are highly regulated in normal cells and in the absence of extracellular stimuli are maintained in an inactive conformation. However, some Src family members, for example c-Src tyrosine kinase is frequently significantly activated (when compared to normal cell levels) in common human cancers such as gastrointestinal cancer, for example colon, rectal and stomach cancer, and breast cancer. The Src family of non-receptor tyrosine kinases has also been located in other common human cancers. It is further known that the predominant role of c-Src non-receptor kinase is to regulate the assembly of focal adhesion complexes through interaction with a number of cytoplasmic proteins including, for example, focal adhesion kinase and paxillin. In addition c-Src is coupled to signaling pathways that regulate the actin cytoskeleton which facilities cell motility. Likewise, important roles are played by the c-Src, c-Yes and c-Fyn non-receptor tyrosine kinases in integrin mediated signaling and In disrupting cadherin-dependent cell-cell junctions. Cellular motility is necessarily required for a localized tumor to progress through the stages of dissemination into the blood stream, invasion of other tissues and initiation of metastatic tumor growth. For example, colon tumor progression from localized to disseminated, invasive metastatic disease has been correlated with c-Src non-receptor tyrosine kinase activity. Accordingly it has been recognized that an inhibitor of such non-receptor tyrosine kinases should be of value as an inhibitor of the motility of tumor cells and as an inhibitor of the dissemination and invasiveness of mammalian cancer cells leading to inhibition of metastatic tumor growth. In particular an inhibitor of such non-receptor tyrosine kinases should be of value as an anti-invasive agent for use in the containment and/or treatment of solid tumor diseases.
The compounds of the present invention possess activities to one or more tyrosine kinases described herein, in particular selected from the group consisting of Tie-2, VEGFR-2, Src-c, and EphB4 proteins, implicated in cancers or atherosclerosis by inhibiting or preventing inappropriate angiogenesis, vasculogenesis, vessel maturation, or cell motilities.